RNA editing is a term used to describe the structural alteration, insertion or deletion of nucleotides in RNA. If the modification occurs in messenger RNA (mRNA), it can result in the translation of a protein sequence different from that predicted by the DNA sequence of the gene. Since the discovery of RNA editing enzymes that act on the mRNA encoded in the nuclei of eukaryotic cells, the gene sequences of higher organisms can no longer be assumed to fully describe the protein sequences. Thus, RNA editing plays a pivotal role in the basic process of information transfer that takes place during protein expression. Moreover, human proteins translated from editing messages (erg. glutamate and serotonin receptors) have been implicated in a number of neurodegenerative, psychiatric and behavior disorders such as stroke, epilepsy, Parkinson's disease and schizophrenia. Human pathogens also use RNA editing to their advantage. Hepatitis delta virus, a subviral human pathogen that increases the chance of severe liver disease during concurrent infection with hepatitis B, requires RNA editing at a stop codon to produce two proteins from the same message. Both proteins are required for proper replication and packaging of hepatitis delta virus. In many cases, an enzyme capable of a particular RNA modification has been identified and cloned. However, our understanding of the molecular basis for the fundamental steps in the editing reactions, such as how the substrate is recognized, is surprisingly limited. The goal of this research is to define mechanistic properties of an RNA editing adenosine deaminase (ADAR-2). This will be accomplished through the synthesis of nonnatural RNA substrate analogs and their detailed analysis using a combination of enzyme kinetics, gel mobility shift experiments and fluorescence measurements. Prior to our work, no experimental system existed for a detailed, systematic analysis of RNA editing mechanisms. The results of these studies will extend our basic understanding of the important process of RNA editing and may ultimately lead to the ability to control the structure of cellular or viral RNAs and the proteins they encode. Furthermore, we aim to develop reagents that can be used in collaboration to elucidate the structure of the ADAR/RNA complex at distinct points along the reaction coordinate.
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